Cerebral ischemia, most notably in the form of stroke, is an ever-growing burden on both the US and world populations. There is currently only 1 clinically approved treatment for stroke - tissue plasminogen activator - a blood clot dissolver that has limited application to a small cohort of patients within a narrow time frame. Many post-stroke therapeutic agents have gone to clinical trial yet none thus far have shown efficacy. Prophylactic treatment is an alternative strategy for neuroprotection that is supported by a large amount of literature demonstrating the therapeutic potential of preconditioning paradigms. The main objective of this proposal is to further provide preclinical evidence for preconditioning with the naturally occurring, polyphenolic small molecule resveratrol. Our pilot study suggests that resveratrol preconditioning (RPC) can induce a chronic ischemic tolerance that lasts for at least 2 weeks in vivo. To further expand this study, we propose the following specific aims to delineate the mechanisms of this chronic ischemic tolerance. In aim 1 we will characterize a novel transgenic knockout mouse model and test the Sirt1 dependence of RPC-induced chronic ischemic tolerance. We are generating tamoxifen-inducible neuron-specific Sirt1 knockout mice which will be subjected to a rodent model of focal cerebral ischemia - middle cerebral artery occlusion (MCAo). Aim 2 will assess the ability of RPC to protect DNA from oxidative damage via Sirt1 by compacting chromatin and enhancing DNA repair pathways, and ultimately inhibiting apoptosis. For these experiments we will employ a DNase I digestion assay both in vivo and ex vivo with organotypic cortical slice cultures exposed to oxygen and glucose deprivation (OGD, in vitro ischemia) followed by quantification of oxidative DNA lesions, repair activity and apoptosis. Mitochondria will be the focus of Aim 3; organotypic cultures will be utilized to investigate a novel pathway for neuroprotection, the mitochondrial unfolfed protein response (UPRmt). Molecular approaches both in vivo and ex vivo will be employed to determine resveratrol-mediated activation of the UPRmt, establish its functional effects and its role in ischemic protection. Taken together, the results of this proposal will add to the body of literature supporting pharmacological preconditioning as a potential neuroprotective strategy, particularly for stroke, and provide strong preclinical evidence for resveratrol as a translatable pharmacologic agent. Such an agent can be administered in a prophylactic manner to a large population at risk for stroke. Furthermore, identification of a long-lasting neuroprotective agent, and its underlying mechanism(s), will have implications not only in several other forms of ischemia, such as cardiac arrest, but also other neurological disorders with similar pathology.